In grassland ecosystems, N and P fertilization often increase plant productivity, but there is no concensus if fertilization affects soil C fractions. We tested effects of N, P and N+P fertilization at 5, 10, 15 g m−2 yr−1 N5, N10, N15, P5, P10, P15, N5P5, N10P10, and N15P15 compared to unfertilized control on soil C, soil microbial biomass and functional diversity at the 0–20 cm and 20–40 cm depth in an alpine meadow after 5 years of continuous fertilization. Fertilization increased total aboveground biomass of community and grass but decreased legume and forb biomass compared to no fertilization. All fertilization treatments decreased the C:N ratios of legumes and roots compared to control, however fertilization at rates of 5 and 15 g m−2 yr−1 decreased the C:N ratios of the grasses. Compared to the control, soil microbial biomass C increased in N5, N10, P5, and P10 in 0–20 cm, and increased in N10 and P5 while decreased in other treatments in 20–40 cm. Most of the fertilization treatments decreased the respiratory quotient qCO2 in 0–20 cm but increased qCO2 in 20–40 cm. Fertilization increased soil microbial functional diversity except N15 but decreased cumulative C mineralization except in N15 in 0–20 cm and N5 in 20–40 cm. Soil organic C SOC decreased in P5 and P15 in 0–20 cm and for most of the fertilization treatments except N15P15 in 20–40 cm. Overall, these results suggested that soils will not be a C sink except N15P15. Nitrogen and phosphorus fertilization may lower the SOC pool by altering the plant biomass composition, especially the C:N ratios of different plant functional groups, and modifying C substrate utilization patterns of soil microbial communities. The N+P fertilization at 15 g m−2 yr−1 may be used in increasing plant aboveground biomass and soil C accumulation under these meadows.